Esma N. Zeydanli

Intracellular free zinc during cardiac excitation- contraction cycle: calcium and redox dependencies

AIMS Zinc exists in biological systems as bound and histochemically reactive free Zn(2+). It is an essential structural constituent of many proteins, including enzymes from cellular signalling pathways, in which it functions as a signalling molecule. In cardiomyocytes at rest, Zn(2+) concentration is in the nanomolar range. Very little is known about precise mechanisms controlling the intracellular distribution of Zn(2+) and its variations during cardiac function. METHODS AND RESULTS Live-cell detection of intracellular Zn(2+) has become feasible through the recent development of Zn(2+)-sensitive and -selective fluorophores able to distinguish Zn(2+) from Ca(2+). Here, in freshly isolated rat cardiomyocytes, we investigated the rapid changes in Zn(2+) homeostasis using the Zn(2+)-specific fluorescent dye, FluoZin-3, in comparison to Ca(2+)-dependent fluo-3 fluorescence. Zn(2+) sparks and Zn(2+) transients, in quiescent and electrically stimulated cardiomyocytes, respectively, were visualized in a similar manner to known rapid Ca(2+) changes. Both Zn(2+) sparks and Zn(2+) transients required Ca(2+) entry. Inhibiting the sarcoplasmic reticulum Ca(2+) release or increasing the Ca(2+) load in a low-Na(+) solution suppressed or increased Zn(2+) movements, respectively. Mitochondrial inhibitors slightly reduced both Zn(2+) sparks and Zn(2+) transients. Oxidation by H₂O₂ facilitated and acidic pH inhibited the Ca(2+)-dependent Zn(2+) release. CONCLUSION It is proposed that Zn(2+) release during the cardiac cycle results mostly from intracellular free Ca(2+) increase, triggering production of reactive oxygen species that induce changes in metal-binding properties of metallothioneins and other redox-active proteins, aside from ionic exchange on these proteins.

Antioxidants but not doxycycline treatments restore depressed beta-adrenergic responses of the heart in diabetic rats.

Reactive oxygen species (ROS) play important roles in the development of diabetic cardiomyopathy. Matrix metalloproteinases (MMPs) can get activated by ROS and contribute to loss of myocardial contractile function in oxidative stress injury. Previously we have shown that either a MMP-2 inhibitor doxycycline or an antioxidant selenium treatment in vivo prevented diabetes-induced cardiac dysfunction significantly. In addition, there is an evidence for impaired cardiac responsiveness to β-adrenoceptor (βAR) stimulation in experimental animals with diabetes. The exact nature of linkage between the functional depression in cardiac responses to catecholamines and the variations in uncoupling of βAR in diabetes has not been clearly defined. Therefore, we aimed to evaluate the effect of in vivo administration of doxycycline on βAR responses of isolated hearts from diabetic rats and compare these data with two well-known antioxidants; sodium selenate and (n−3) fatty acid-treated diabetic rats. We examined the changes in the basal cardiac function in response to the βAR stimulation, adenylate cyclase activity, and βAR affinity to its agonist, isoproterenol. These results showed that antioxidant treatment of diabetic rats could protect the hearts against diabetes-induced depression in βAR responses, significantly while doxycycline did not have any significant beneficial action on these parameters. As a summary, present data, in part, demonstrate that antioxidants and MMP inhibitors could both regulate MMP function but may also utilize different mechanisms of action in cardiomyocytes, particularly related with βAR signaling pathway.

Age-related regulation of excitation–contraction coupling in rat heart

Hearts from subjects with different ages have different Ca2+ signaling. Release of Ca2+ from intracellular stores in response to an action potential initiates cardiac contraction. Both depolarization-stimulated and spontaneous Ca2+ releases, Ca2+ transients and Ca2+ sparks, demonstrate the main events of excitation–contraction coupling (ECC). Global increase in free Ca2+ concentration ([Ca2+]i) consists of summation of Ca2+ release events in cardiomyocytes. Since the Ca2+ flux induced by Ca2+ sparks reports a summation of ryanodine-sensitive Ca2+ release channels (RyR2s)’s behavior in a spark cluster, evaluation of the properties of Ca2+ sparks and Ca2+ transients may provide insight into the role of RyR2s on altered heart function between 3-month-old (young adult) and 6-month-old (mature adult) rats. Basal [Ca2+]i and Ca2+ sparks frequency were significantly higher in mature adult rats compared to those of young adults. Moreover, amplitudes of Ca2+ sparks and Ca2+ transients were significantly smaller in mature adults than those of young adults with longer time courses. A smaller L-type Ca2+ current density and decreased SR Ca2+ load was observed in mature adult rats. In addition, RyR2s were markedly hyperphosphorylated, and phosphorylation levels of PKA and CaMKII were higher in heart from mature adults compared to those of young adults, whereas their SERCA protein levels were similar. Our data demonstrate that hearts from rats with different ages have different Ca2+ signaling including hyperphosphorylation of RyR2s and higher basal [Ca2+]i together with increased oxidized protein-thiols in mature adult rats compared to those of young adults, which play important roles in ECC. Finally, we report that ECC efficiency changes with age during maturation, partially related with an increased cellular oxidation level leading to reduced free protein-thiols in cardiomyocytes.

Beneficial effect of sodium selenate on vascular dysfunction in diabetic rats

Since selenium compounds can restore some metabolic parameters and structural alterations of diabetic rat tissues, we tempted to investigate whether these beneficial effects extend to the diabetic rat isolated thoracic aorta dysfunctions. Diabetes was induced by streptozotocin (50 mg/kg body weight) and rats were treated with sodium selenate (15 μmol/kg body weight/ day) for 4 weeks. Sodium selenate treatment of diabetic rats induced a significant recovery (80% wrt diabetes) in the depressed phenylephrine (10–10 M) stimulated isometric contractions (50% wrt control) in aorta while we obtained 100% recovery in the depressed relaxation responses (30% wrt control) with isoproterenol (10–10 M) without any significant changes in Log50 values. Sodium selenate treatment of the diabetic rats also restored the altered activities of several antioxidants enzymes of which are involved in the glutathione metabolism of the heart as well as the levels of glutathione and oxidized protein sulphydryls while no significant effect on high blood glucose level. Our data indicate that an oxidant shift of cellular thiolic pools can modulate contraction-relaxation activities of thoracic aorta in diabetic rats. Therefore, it can be summarized that selenium employs important roles in altered contraction-relaxation activities of thoracic aorta via affecting the glutathione redox cycle to combat oxidative stress in diabetes and small doses of selenium compounds may be useful as an adjunctive therapy in human diabetes.